An official website of the United States government
Abstract Di‐ubiquitin (diUB) conjugates of defined linkages are useful tools for probing the functions of UB ligases, UB‐binding proteins and deubiquitinating enzymes (DUBs) in coding, decoding and editing the signals carried by the UB chains. Here we developed an efficient method for linkage‐specific synthesis of diUB probes based on the incorporation of the unnatural amino acid (UAA)Nϵ‐L‐thiaprolyl‐L‐Lys (L‐ThzK) into UB for ligation with another UB at a defined Lys position. The diUB formed by the UAA‐mediated ligation reaction has a G76C mutation on the side of donor UB for conjugation with E2 and E3 enzymes or undergoing dethiolation to generate a covalent trap for DUBs. The development of UAA mutagenesis for diUB synthesis provides an easy route for preparing linkage‐specific UB‐based probes to decipher the biological signals mediated by protein ubiquitination.
more »
« less
UbE3-APA: a bioinformatic strategy to elucidate ubiquitin E3 ligase activities in quantitative proteomics study
Abstract MotivationUbiquitination is widely involved in protein homeostasis and cell signaling. Ubiquitin E3 ligases are critical regulators of ubiquitination that recognize and recruit specific ubiquitination targets for the final rate-limiting step of ubiquitin transfer reactions. Understanding the ubiquitin E3 ligase activities will provide knowledge in the upstream regulator of the ubiquitination pathway and reveal potential mechanisms in biological processes and disease progression. Recent advances in mass spectrometry-based proteomics have enabled deep profiling of ubiquitylome in a quantitative manner. Yet, functional analysis of ubiquitylome dynamics and pathway activity remains challenging. ResultsHere, we developed a UbE3-APA, a computational algorithm and stand-alone python-based software for Ub E3 ligase Activity Profiling Analysis. Combining an integrated annotation database with statistical analysis, UbE3-APA identifies significantly activated or suppressed E3 ligases based on quantitative ubiquitylome proteomics datasets. Benchmarking the software with published quantitative ubiquitylome analysis confirms the genetic manipulation of SPOP enzyme activity through overexpression and mutation. Application of the algorithm in the re-analysis of a large cohort of ubiquitination proteomics study revealed the activation of PARKIN and the co-activation of other E3 ligases in mitochondria depolarization-induced mitophagy process. We further demonstrated the application of the algorithm in the DIA (data-independent acquisition)-based quantitative ubiquitylome analysis. Availability and implementationSource code and binaries are freely available for download at URL: https://github.com/Chenlab-UMN/Ub-E3-ligase-Activity-Profiling-Analysis, implemented in python and supported on Linux and MS Windows. Supplementary informationSupplementary data are available at Bioinformatics online.
more »
« less
- Award ID(s):
- 1753154
- PAR ID:
- 10394807
- Publisher / Repository:
- Oxford University Press
- Date Published:
- Journal Name:
- Bioinformatics
- Volume:
- 38
- Issue:
- 8
- ISSN:
- 1367-4803
- Page Range / eLocation ID:
- p. 2211-2218
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
More Like this
-
-
The time-dependent degradation of core circadian clock proteins is essential for the proper functioning of circadian timekeeping mechanisms that drive daily rhythms in gene expression and, ultimately, an organism’s physiology. The ubiquitin proteasome system plays a critical role in regulating the stability of most proteins, including the core clock components. Our laboratory developed a cell-based functional screen to identify ubiquitin ligases that degrade any protein of interest and have started screening for those ligases that degrade circadian clock proteins. This screen identified Spsb4 as a putative novel E3 ligase for RevErbα. In this article, we further investigate the role of Spsb4 and its paralogs in RevErbα stability and circadian rhythmicity. Our results indicate that the paralogs Spsb1 and Spsb4, but not Spsb2 and Spsb3, can interact with and facilitate RevErbα ubiquitination and degradation and regulate circadian clock periodicity.more » « less
-
null (Ed.)Glycosyltransferase OGT catalyzes the conjugation of O-linked β-D-N-acetylglucosamine (O-GlcNAc) to Ser and Thr residues of the cellular proteins and regulates many key processes in the cell. Here, we report the identification of OGT as a ubiquitination target of HECT-type E3 ubiquitin (UB) ligase E6AP, whose overexpression in HEK293 cells would induce the degradation of OGT. We also found that the expression of E6AP in HeLa cells with the endogenous expression of the E6 protein of the human papillomavirus (HPV) would accelerate OGT degradation by the proteasome and suppress O-GlcNAc modification of OGT substrates in the cell. Overall, our study establishes a new mechanism of OGT regulation by the ubiquitin–proteasome system (UPS) that mediates the crosstalk between protein ubiquitination and O-GlcNAcylation pathways underlying diverse cellular processes.more » « less
-
Abstract Target validation is key to the development of protein degrading molecules such as proteolysis‐targeting chimeras (PROTACs) to identify cellular proteins amenable for induced degradation by the ubiquitin‐proteasome system (UPS). Previously the HaloPROTAC system was developed to screen targets of PROTACs by linking the chlorohexyl group with the ligands of E3 ubiquitin ligases VHL and cIAP1 to recruit target proteins fused to the HaloTag for E3‐catalyzed ubiquitination. Reported here are HaloPROTACs that engage the cereblon (CRBN) E3 to ubiquitinate and degrade HaloTagged proteins. A focused library of CRBN‐pairing HaloPROTACs was synthesized and screened to identify efficient degraders of EGFP‐HaloTag fusion with higher activities than VHL‐engaging HaloPROTACs at sub‐micromolar concentrations of the compound. The CRBN‐engaging HaloPROTACs broadens the scope of the E3 ubiquitin ligases that can be utilized to screen suitable targets for induced protein degradation in the cell.more » « less
-
Generating new strategies to improve plant performance and yield in crop plants becomes increasingly relevant with ongoing and predicted global climate changes. E3 ligases that function as key regulators within the ubiquitin proteasome pathway often are involved in abiotic stress responses, development, and metabolism in plants. The aim of this research was to transiently downregulate an E3 ligase that uses BTB/POZ-MATH proteins as substrate adaptors in a tissue-specific manner. Interfering with the E3 ligase at the seedling stage and in developing seeds results in increased salt-stress tolerance and elevated fatty acid levels, respectively. This novel approach can help to improve specific traits in crop plants to maintain sustainable agriculture.more » « less
